Effects of Environmental and Water Quality Variables on Histamine-Producing Bacteria Concentration and Species in the Northern Gulf of Mexico.

Scombrotoxin (histamine) fish poisoning is a common seafood-borne illness attributed to toxin production by histamine-producing bacteria (HPB) in fish tissues during decomposition. In laboratory studies, growth of HPB and other bacterial species is affected by physical and chemical attributes, but natural communities of HPB are not well understood. To determine how in situ environmental and water quality variables may affect density of HPB in the natural aquatic environment, we compared presence and abundance of HPB to ambient temperature, salinity, dissolved oxygen, fecal coliforms, male-specific coliphage, nutrient concentrations, carbon and nitrogen stable isotope ratios, and C:N in water samples collected from July 2017 to February 2018 along a natural salinity gradient in a tidal river on the coast of northern Gulf of Mexico. HPB in water samples were quantified using a real-time PCR, most probable number method. HPB species were identified via 16S rRNA gene sequences. Temperature and salinity were determined to be the main factors driving HPB presence and concentration. Canonical correspondence analysis revealed that different HPB were associated with different environmental conditions. Photobacterium damselae was found under warmer, higher-salinity conditions; Raoultella planticola was found at colder, lower-salinity conditions; Enterobacter aerogenes was found at warmer, lower-salinity conditions; and Morganella morganii was found at most sites, independent of environmental conditions. These results showed that naturally occurring HPB abundance and species composition can be affected by environmental conditions, which could manifest in various potentials for histamine formation and scombrotoxin fish poisoning risk based on environmental factors. IMPORTANCE This study determined the effects of environmental conditions on presence and abundance of naturally occurring histamine-producing bacteria in the northern Gulf of Mexico. Here, we show that HPB abundance and species composition are related to in situ ambient temperature and salinity, with the magnitude of this effect dependent on the particular HPB species. This finding suggests that environmental conditions at fishing sites could affect the risk of human illness from scombrotoxin (histamine) fish poisoning.

Genomic Complexity Predicts Resistance to Endocrine Therapy and CDK4/6 Inhibition in Hormone Receptor-Positive (HR+)/HER2-Negative Metastatic Breast Cancer.

PURPOSE: Clinical biomarkers to identify patients unlikely to benefit from CDK4/6 inhibition (CDK4/6i) in combination with endocrine therapy (ET) are lacking. We implemented a comprehensive circulating tumor DNA (ctDNA) analysis to identify genomic features for predicting and monitoring treatment resistance. EXPERIMENTAL DESIGN: ctDNA was isolated from 216 plasma samples collected from 51 patients with hormone receptor-positive (HR+)/HER2-negative (HER2-) metastatic breast cancer (MBC) on a phase II trial of palbociclib combined with letrozole or fulvestrant (NCT03007979). Boosted whole-exome sequencing (WES) was performed at baseline and clinical progression to evaluate genomic alterations, mutational signatures, and blood tumor mutational burden (bTMB). Low-pass whole-genome sequencing was performed at baseline and serial timepoints to assess blood copy-number burden (bCNB). RESULTS: High bTMB and bCNB were associated with lack of clinical benefit and significantly shorter progression-free survival (PFS) compared with patients with low bTMB or low bCNB (all P < 0.05). Dominant APOBEC signatures were detected at baseline exclusively in cases with high bTMB (5/13, 38.5%) versus low bTMB (0/37, 0%; P = 0.0006). Alterations in ESR1 were enriched in samples with high bTMB (P = 0.0005). There was a high correlation between bTMB determined by WES and bTMB determined using a 600-gene panel (R = 0.98). During serial monitoring, an increase in bCNB score preceded radiographic progression in 12 of 18 (66.7%) patients. CONCLUSIONS: Genomic complexity detected by noninvasive profiling of bTMB and bCNB predicted poor outcomes in patients treated with ET and CDK4/6i and identified early disease progression before imaging. Novel treatment strategies including immunotherapy-based combinations should be investigated in this population.

A phase II study of palbociclib plus letrozole plus trastuzumab as neoadjuvant treatment for clinical stages II and III ER+ HER2+ breast cancer (PALTAN).

Patients with ER+/HER2+ breast cancer (BC) are less likely to achieve pathological complete response (pCR) after chemotherapy with dual HER2 blockade than ER-/HER2+ BC. Endocrine therapy plus trastuzumab is effective in advanced ER+/HER2+ BC. Inhibition of CDK4/6 and HER2 results in synergistic cell proliferation reduction. We combined palbociclib, letrozole, and trastuzumab (PLT) as a chemotherapy-sparing regimen. We evaluated neoadjuvant PLT in early ER+/HER2+ BC. Primary endpoint was pCR after 16 weeks. Research biopsies were performed for whole exome and RNA sequencing, PAM50 subtyping, and Ki67 assessment for complete cell cycle arrest (CCCA: Ki67 ≤ 2.7%). After 26 patients, accrual stopped due to futility. pCR (residual cancer burden-RCB 0) was 7.7%, RCB 0/I was 38.5%. Grade (G) 3/4 treatment-emergent adverse events occurred in 19. Among these, G3/4 neutropenia was 50%, hypertension 26.9%, and leucopenia 7.7%. Analysis indicated CCCA in 85% at C1 day 15 (C1D15), compared to 27% at surgery after palbociclib was discontinued. Baseline PAM50 subtyping identified 31.2% HER2-E, 43.8% Luminal B, and 25% Luminal A. 161 genes were differentially expressed comparing C1D15 to baseline. MKI67, TK1, CCNB1, AURKB, and PLK1 were among the genes downregulated, consistent with CCCA at C1D15. Molecular Signatures Database gene-sets analyses demonstrated downregulated processes involved in proliferation, ER and mTORC1 signaling, and DNA damage repair at C1D15, consistent with the study drug's mechanisms of action. Neoadjuvant PLT showed a pCR of 7.7% and an RCB 0/I rate of 38.5%. RNA sequencing and Ki67 data indicated potent anti-proliferative effects of study treatments. ClinicalTrials.gov- NCT02907918.

A phase II trial of an alternative schedule of palbociclib and embedded serum TK1 analysis.

Palbociclib 3-weeks-on/1-week-off, combined with hormonal therapy, is approved for hormone receptor positive (HR+)/HER2-negative (HER2-) advanced/metastatic breast cancer (MBC). Neutropenia is the most frequent adverse event (AE). We aim to determine whether an alternative 5-days-on/2-days-off weekly schedule reduces grade 3 and above neutropenia (G3 + ANC) incidence. In this single-arm phase II trial, patients with HR+/HER2- MBC received palbociclib 125 mg, 5-days-on/2-days-off, plus letrozole or fulvestrant per physician, on a 28-day cycle (C), as their first- or second-line treatment. The primary endpoint was G3 + ANC in the first 29 days (C1). Secondary endpoints included AEs, efficacy, and serum thymidine kinase 1 (sTK1) activity. At data-cutoff, fifty-four patients received a median of 13 cycles (range 2.6-43.5). The rate of G3 + ANC was 21.3% (95% CI: 11.2-36.1%) without G4 in C1, and 40.7% (95% CI: 27.9-54.9%), including 38.9% G3 and 1.8% G4, in all cycles. The clinical benefit rate was 80.4% (95% CI: 66.5-89.7%). The median progression-free survival (mPFS) (95% CI) was 19.75 (12.11-34.89), 33.5 (17.25-not reached [NR]), and 11.96 (10.43-NR) months, in the overall, endocrine sensitive or resistant population, respectively. High sTK1 at baseline, C1 day 15 (C1D15), and C2D1 were independently prognostic for shorter PFS (p = 9.91 × 10-4, 0.001, 0.007, respectively). sTK1 decreased on C1D15 (p = 4.03 × 10-7), indicating target inhibition. Rise in sTK1 predicted progression, with the median lead time of 59.5 (inter-quartile range: -206.25-0) days. Palbociclib, 5-days-on/2-days-off weekly, met its primary endpoint with reduced G3 + ANC, without compromising efficacy. sTK1 is prognostic and shows promise in monitoring the palbociclib response. ClinicalTrials.gov#: NCT3007979.

Immunogenomic profiling and pathological response results from a clinical trial of docetaxel and carboplatin in triple-negative breast cancer.

PURPOSE: Patients with triple-negative breast cancer (TNBC) who do not achieve pathological complete response (pCR) following neoadjuvant chemotherapy have a high risk of recurrence and death. Molecular characterization may identify patients unlikely to achieve pCR. This neoadjuvant trial was conducted to determine the pCR rate with docetaxel and carboplatin and to identify molecular alterations and/or immune gene signatures predicting pCR. EXPERIMENTAL DESIGN: Patients with clinical stages II/III TNBC received 6 cycles of docetaxel and carboplatin. The primary objective was to determine if neoadjuvant docetaxel and carboplatin would increase the pCR rate in TNBC compared to historical expectations. We performed whole-exome sequencing (WES) and immune profiling on pre-treatment tumor samples to identify alterations that may predict pCR. Thirteen matching on-treatment samples were also analyzed to assess changes in molecular profiles. RESULTS: Fifty-eight of 127 (45.7%) patients achieved pCR. There was a non-significant trend toward higher mutation burden for patients with residual cancer burden (RCB) 0/I versus RCB II/III (median 80 versus 68 variants, p 0.88). TP53 was the most frequently mutated gene, observed in 85.7% of tumors. EGFR, RB1, RAD51AP2, SDK2, L1CAM, KPRP, PCDHA1, CACNA1S, CFAP58, COL22A1, and COL4A5 mutations were observed almost exclusively in pre-treatment samples from patients who achieved pCR. Seven mutations in PCDHA1 were observed in pre-treatment samples from patients who did not achieve pCR. Several immune gene signatures including IDO1, PD-L1, interferon gamma signaling, CTLA4, cytotoxicity, tumor inflammation signature, inflammatory chemokines, cytotoxic cells, lymphoid, PD-L2, exhausted CD8, Tregs, and immunoproteasome were upregulated in pre-treatment samples from patients who achieved pCR. CONCLUSION: Neoadjuvant docetaxel and carboplatin resulted in a pCR of 45.7%. WES and immune profiling differentiated patients with and without pCR. TRIAL REGISTRATION: Clinical trial information: NCT02124902, Registered 24 April 2014 & NCT02547987, Registered 10 September 2015.

Early Assessment Window for Predicting Breast Cancer Neoadjuvant Therapy using Biomarkers, Ultrasound, and Diffuse Optical Tomography.

PURPOSE: The purpose of the study was to assess the utility of tumor biomarkers, ultrasound (US) and US-guided diffuse optical tomography (DOT) in early prediction of breast cancer response to neoadjuvant therapy (NAT). METHODS: This prospective HIPAA compliant study was approved by the institutional review board. Forty one patients were imaged with US and US-guided DOT prior to NAT, at completion of the first three treatment cycles, and prior to definitive surgery from February 2017 to January 2020. Miller-Payne grading was used to assess pathologic response. Receiver operating characteristic curves (ROCs) were derived from logistic regression using independent variables, including: tumor biomarkers, US maximum diameter, percentage reduction of the diameter (%US), pretreatment maximum total hemoglobin concentration (HbT) and percentage reduction in HbT (%HbT) at different treatment time points. Resulting ROCs were compared using area under the curve (AUC). Statistical significance was tested using two-sided two-sample student t-test with P < 0.05 considered statistically significant. Logistic regression was used for ROC analysis. RESULTS: Thirty-eight patients (mean age = 47, range 24-71 years) successfully completed the study, including 15 HER2 + of which 11 were ER + ; 12 ER + or PR + /HER2-, and 11 triple negative. The combination of HER2 and ER biomarkers, %HbT at the end of cycle 1 (EOC1) and %US (EOC1) provided the best early prediction, AUC = 0.941 (95% CI 0.869-1.0). Similarly an AUC of 0.910 (95% CI 0.810-1.0) with %US (EOC1) and %HbT (EOC1) can be achieved independent of HER2 and ER status. The most accurate prediction, AUC = 0.974 (95% CI 0.933-1.0), was achieved with %US at EOC1 and %HbT (EOC3) independent of biomarker status. CONCLUSION: The combined use of tumor HER2 and ER status, US, and US-guided DOT may provide accurate prediction of NAT response as early as the completion of the first treatment cycle. CLINICAL TRIAL REGISTRATION NUMBER: NCT02891681. https://clinicaltrials.gov/ct2/show/NCT02891681 , Registration time: September 7, 2016.

Interim Analysis of the Phase II Study: Noninferiority Study of Doxorubicin with Upfront Dexrazoxane plus Olaratumab for Advanced or Metastatic Soft-Tissue Sarcoma.

PURPOSE: To report the interim analysis of the phase II single-arm noninferiority trial, testing the upfront use of dexrazoxane with doxorubicin on progression-free survival (PFS) and cardiac function in soft-tissue sarcoma (STS). PATIENTS AND METHODS: Patients with metastatic or unresectable STS who were candidates for first-line treatment with doxorubicin were deemed eligible. An interim analysis was initiated after 33 of 65 patients were enrolled. Using the historical control of 4.6 months PFS for doxorubicin in the front-line setting, we tested whether the addition of dexrazoxane affected the efficacy of doxorubicin in STS. The study was powered so that a decrease of PFS to 3.7 months would be considered noninferior. Secondary aims included cardiac-related mortality, incidence of heart failure/cardiomyopathy, and expansion of cardiac monitoring parameters including three-dimensional echocardiography. Patients were allowed to continue on doxorubicin beyond 600 mg/m2 if they were deriving benefit and were not demonstrating evidence of symptomatic cardiac dysfunction. RESULTS: At interim analysis, upfront use of dexrazoxane with doxorubicin demonstrated a PFS of 8.4 months (95% confidence interval: 5.1-11.2 months). Only 3 patients were removed from study for cardiotoxicity, all on > 600 mg/m2 doxorubicin. No patients required cardiac hospitalization or had new, persistent cardiac dysfunction with left ventricular ejection fraction remaining below 50%. The median administered doxorubicin dose was 450 mg/m2 (interquartile range, 300-750 mg/m2). CONCLUSIONS: At interim analysis, dexrazoxane did not reduce PFS in patients with STS treated with doxorubicin. Involvement of cardio-oncologists is beneficial for the monitoring and safe use of high-dose anthracyclines in STS.See related commentary by Benjamin and Minotti, p. 3809.

Mutation profile differences in younger and older patients with advanced breast cancer using circulating tumor DNA (ctDNA).

PURPOSE: Little is known regarding the mutation profiles of ctDNA in the older adult breast cancer population. The objective of this study is to assess differences in mutation profiles in the older adult breast cancer population using a ctDNA assay as well as assess utilization of testing results. METHODS: Patients with advanced breast cancer underwent molecular profiling using a plasma-based ctDNA NGS assay (Guardant360) between 5/2015 and 10/2019 at Siteman Cancer Center. The profiling results of a multi-institutional database of patients with advanced breast cancer who had undergone molecular profiling were obtained. Associations between mutations and age group (≥ 65 vs. < 65) were examined using a Fisher's exact test. RESULTS: In the single-institutional cohort, 148 patients (69.2%) were < 65 years old and 66 patients (30.8%) ≥ 65 years old. ATM, BRAF, and PIK3CA mutations were found more frequently in older patients with ER + HER2- breast cancers (p < 0.01). In the multi-institutional cohort, 5367 (61.1%) were < 65 years old and 3417 (38.9%) ≥ 65 years old. ATM, PIK3CA, and TP53 mutations were more common in the older cohort (p < 0.0001) and MYC and GATA3 mutations were less common in the older cohort (p < 0.0001). CtDNA testing influenced next-line treatment management in 40 (19.8%) patients in the single-institutional cohort. CONCLUSION: When controlling for subtype, results from a single institution were similar to the multi-institutional cohort showing that ATM and PIK3CA were more common in older adults. These data suggest there may be additional molecular differences in older adults with advanced breast cancers.

Randomized controlled trial of high-dose versus standard-dose vitamin D3 for prevention of aromatase inhibitor-induced arthralgia.

PURPOSE: Half of hormone receptor-positive (HR+) breast cancer patients will develop joint pain, termed aromatase inhibitor-induced arthralgia (AIA), while taking aromatase inhibitor therapy. Though there is no universally accepted effective treatment for AIA, there has been some evidence to support high-dose vitamin D as a treatment. METHODS: We randomized post-menopausal women who were beginning adjuvant AI therapy to receive standard-dose vitamin D3 (800 IU daily for 52 weeks), or high-dose vitamin D3 (50,000 IU weekly for 12 weeks, followed by 2000 IU daily for 40 weeks). The primary end point was development of AIA. The trial was designed to enroll 184 patients. This futility analysis was performed after 93 patients were enrolled. RESULTS: The high-dose vitamin D regimen was effective in raising serum vitamin D levels, but there was no significant difference in development of AIA between the two arms. In the high-dose arm, 25 patients (54%) developed AIA, compared to 27 patients (57%) in the standard-dose arm. The planned futility analysis was positive; thus, the study was terminated. Neither baseline vitamin D nor 12-week vitamin D level was predictive of AIA development. CONCLUSION: Although vitamin D levels were increased in the high-dose arm, there was no significant signal for benefit of high-dose vitamin D supplementation for AIA prevention in this unblinded trial. This study, along with several others, implies that vitamin D likely does not play a significant role in AIA for the majority of patients.

Retrospective Analysis of Treatment Patterns and Effectiveness of Palbociclib and Subsequent Regimens in Metastatic Breast Cancer.

Background: Cyclin-dependent kinase (CDK) 4/6 inhibitors are now the standard of care for hormone receptor-positive (HR+), HER2-negative (HER-) metastatic breast cancer (MBC). However, guidelines are lacking regarding their optimal sequencing with other available agents. This study examines physician practice patterns and treatment outcomes of palbociclib and subsequent therapies in a real-world setting. Methods: A retrospective chart review was conducted for consecutive patients with MBC who received palbociclib between February 2015 and August 2017 at the Alvin J. Siteman Cancer Center. Kaplan-Meier method was used to generate time-to-event curves and estimate median progression-free survival (mPFS). Log-rank test was used to compare differences. Results: A total of 200 patients, with a median age of 59.4 years and a follow-up of 19.5 months, were included. Palbociclib was most frequently combined with letrozole (73.5%), followed by fulvestrant (25%), anastrozole (1%), and tamoxifen (0.5%). Most patients received palbociclib in the endocrine-resistant setting (n=42, n=50, and n=108 in the first-, second-, and subsequent-line settings, respectively), and the fraction of patients receiving palbociclib as first- or second-line therapy increased in recent months (P=.0428). mPFS was 20.7, 12.8, and 4.0 months with palbociclib administered in the first-, second-, and subsequent-line settings, respectively (P<.0001). Incidences of grade 3/4 neutropenia (41.5%) and dose reductions (29%) were comparable to reports in the literature. Among patients whose disease progressed on palbociclib (n=104), the most frequent next-line treatment was capecitabine (n=21), followed by eribulin (n=16), nab-paclitaxel (n=15), and exemestane + everolimus (n=12). mPFS with hormone therapy alone or in combination with targeted agents (n=32) after first-, second-, and subsequent-line palbociclib was 17.0, 9.3, and 4.2 months, respectively (P=.04). mPFS with chemotherapy (n=70) was not reached, 4.7, and 4.1 months after first-, second-, and subsequent-line palbociclib, respectively (P=.56). Conclusions: Palbociclib is effective for HR+/HER2- MBC in real-world practice. Hormone therapy alone or in combination with targeted agents remains an effective option after palbociclib progression.

Novel pathways and molecular targets for the treatment of sarcoma.

Sarcomas collectively represent over 100 different subtypes of bone and soft tissue tumors of mesenchymal origin. The low response rate to cytotoxic chemotherapies has necessitated the need for development of either histologically driven or pathway-specific targeted therapies. As our understanding of the molecular mechanisms driving certain subtypes is rapidly advancing, the number of targeted therapies is also increasing. Recently identified novel druggable targets include the MDM2 amplifications in well-differentiated and dedifferentiated liposarcomas, the new translocation NAB2:STAT6 of solitary fibrous tumors, the angiopoeitin-TIE2 pathway in angiosarcoma, the suppression of Mcl1 in X:18/synovial sarcomas, the mTOR pathway in malignant peripheral nerve sheath tumors, CDK4 in alveolar rhabdomyosarcoma, cMET regulation in alveolar soft parts sarcoma, the metabolic abnormalities in wild-type/SHD GIST, and the lack of argininosuccinate synthetase 1 expression seen in most sarcomas. It is through a fundamental understanding of sarcoma biology that clinical trials based on molecular targets can be developed.